The naturally occurring staphylokinase (Sak) is a proteolytic enzyme produced by the lysogenic phage: of Staphylococcus aureus, and consists of 136 amino acid residues. Indeed, Sak is not an enzyme in nature, but it forms a 1:1 complex with plasminogen (plg); in human plasma, which complex is then activated into Sak plm by the trace of plasmin (plm) on the surfaces of blood clots. Sak plm is a potent plasminogen activator to activate the free form of plg into plm, which in turn catalyzes the degradation of fibrin, the main matrix of thrombus, thus resulting in the lysis of thrombus. Sak has fibrin specificity in plg activation and acts more efficiently than other thrombolytic agents to lyse old thrombus and platelet-rich thrombus. Thus, Sak is an efficient and specific thrombolytic agent (Collen D et al, Nature Medicine 4, 279-284 (1998)). At present, recombinant staphylokinases are produced by several companies in the world, but they differ from each other in gene structures. The thrombolytic therapy of acute myocardial infarction (AMI) with recombinant staphylokinases studied by D. Collen of Belgium completed the clinical trial stage II. ShiXin Centre (Chengdu, China) also finished clinical trial stage I of AMI and the effect was quite good. In 1994 Shanghai Medical University constructed a Sak gene, accomplished the high level expression in E. coli, ad finished the pilot process. They have applied for the permission of clinical trials to treat acute cerebral infarction. However, as a heterologous protein, Sak may have strong antigenicity when administered to patients. Though no severe allergic reaction was reported in the clinical trials, Sak induced a high titer of neutralizing antibodies in most patients two weeks after administration, arguing against its repeated administration (Declerck P J et al, Thromb Haemost 71, 129-133 (1994)). Moreover, it was discovered in the study of recombinant staphylokinases that staphylokinases tend to form dimers, even polymers. The formation of polymers increases its immunogenicity.
During the thrombolytic therapy, thrombolytic drugs, are often combined with anti-thrombin or anti-platelet drugs such as heparin and aspirin to promote thrombolysis and to prevent reinfarction. Recent studies of thrombolytic auxiliary drugs are remarkable. Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) are functional sequences against platelet aggregation. They competitively bind to the glycoprotein membrane receptor IIb/IIIa associated with the platelet membrane aggregation, thus preventing the binding of fibrinogen to its receptor and blocking the reformation of thrombus (Frishman W H et al, Am. Heart J. 130, 877-892(1995); Nichols A J et al, Trends Pharmacol. Sci 13, 413-417(1992)). Introducing the RGD/KGD sequence into the cDNA of a thrombolytic agent e.g. urokinase under an appropriate conformational restriction, the product expressed will have the bifunctionality of thromboytics and anticoagulant (Smith J et al, J. Biol. Chem. 270, 30486-30490 (1995)). However, it was indicated in the clinical trials that the thrombolytic effect of urokinase was significantly lower than staphylokinase (50% versus 75%). Furthermore, various chemical mimics have been developed based on the RGD/KGD sequence such as Tirofiban, Lamifiban, Lefradafiban, Orbofiban, Xemilofiban, Integrinlin and the like, which could block the IIb/IIIa receptor. When administered in combination with thrombolytics, the incidence of reinfarction would be significantly decreased (Frishman W H et al, Am. Heart J. 130, 877-892 (1995); Verstraete M et al, 49, 856-884 (1995)).
The object of the invention is to provide novel staphylokinase derivatives, which escape from forming dimer and have the bifunctionality of thrombolytics and anticoagulant, and the preparation thereof.